Phosphate buffer in HPLC

Hi Anto,

Are you certain that the high pressure is coming from the column?  There could just be a blockage in the needle/needle seat.

Hi Abdur,

nice to ear you!

I’m quite sure that the problem is in the column. Here what I did before say thist statement:

-when I saw that the pressure was getting higher and higher, after had washed the column, I’ve change the frit and the golden seal cap

-after that I had a problem with the purge valve: it leaked but the pressure was really high (500bar)

-I’ve removed the column and put the union part: with a 1 ml/min flow 100% MeOH the pressure was 5-6 bar

- I’ve switched several times mainpass-bypass: no differences

- then I tried to wash the column, first of all with milliQ water 50°C, then I change the flow of the column and wash all the system for all weekend long with 95% h2O-5% ACN 0.1 ml/min, the pressure fallen down from 300 bar to 120bar

- I did all the washes with no collection from column to flow cell

Now the instrument is working with another column (Agilent C18 zorbax) with ACN and 0.1% formic acid: with 0.5 ml/min flow is 30 bar, chromatogram is ok

What do you suggest to me?

1. I’ve ordered another column, but I’m so sad.. and think my boss isn’t so happy for this disaster L

THANK YOU

Hi Abdur,

nice to ear you!

I’m quite sure that the problem is in the column. Here what I did before say thist statement:

-when I saw that the pressure was getting higher and higher, after had washed the column, I’ve change the frit and the golden seal cap

-after that I had a problem with the purge valve: it leaked but the pressure was really high (500bar)

-I’ve removed the column and put the union part: with a 1 ml/min flow 100% MeOH the pressure was 5-6 bar

- I’ve switched several times mainpass-bypass: no differences

- then I tried to wash the column, first of all with milliQ water 50°C, then I change the flow of the column and wash all the system for all weekend long with 95% h2O-5% ACN 0.1 ml/min, the pressure fallen down from 300 bar to 120bar

- I did all the washes with no collection from column to flow cell

Now the instrument is working with another column (Agilent C18 zorbax) with ACN and 0.1% formic acid: with 0.5 ml/min flow is 30 bar, chromatogram is ok

What do you suggest to me?

1. I’ve ordered another column, but I’m so sad.. and think my boss isn’t so happy for this disaster L

THANK YOU

Hi Anto,

After preparing your phosphate buffer, do you then filter the buffer before using it?

As long as the buffer is continuously moving in the system you should be ok.  Then if you need to turn the system off, you should make sure to wash with a mixture of water.

You can try to reverse the flow throw the column, by flipping the column in the other direction to see if what is in there can come out the other end.

You might also try looking into a guard column. 

Yes, I filter the buffer with 0.45um sartorius filter and i prepar it fresh every day.

I reverse the flow by changing the direction of the column and let wash for 36 h in 95% water and then by gradient to 100% acn but the pressure still remain high

How is the best way to wash if I have to turn off the instrument?

thanks!

Hi! It seems the column with 2.7 um particles has same good old 2 um frits, just as most 5 um columns:

see page 5:

https://www.agilent.com/cs/library/slidepresentation/public/Short%20OMC%20for%20Tour%20Final.pdf

and page 29:

https://www.agilent.com/cs/library/slidepresentation/public/GCC_2014_Tips_and_Tricks_HPLC_User_Maintenance.pdf

So it is most likely not a solution, as I did filtered with both 0.45 um and 0.2 um when I had issues with this method and it was no difference in clogging.

This may be the problem - 0.45µ filters are not quite adequate for sub 5µ column packings (which you are using).

Try 0.22µ filters for the buffer and samples. This should at least slow down the clogging of your next column.

Also, when you clean out your columns with 5% ACN in water, do you turn them around so flow is backwards? This helps speed up the cleaning process sometimes.

Hi,

yes I’ve change the direction of the column but no news.. 

Antonella Galeone

Quality Control Lab Assistant

Oh, really 0.45um are not enough? 

Sure I’ll try with 0.22 um!

I’m sorry, i’m not so good in english, what do you mean with “do you turn them around so flow is backwards”? Maybe if I do a gradient with the mobile phase?

Thank you!

Antonella Galeone

Quality Control Lab Assistant